: This is an application to study an experimental model for paroxysmal nocturnal hemoglobinuria (PNH). PNH is an acquired stem cell disorder in which a somatic mutation in a bone marrow stem cell disrupts glycoinositol phospholipid (GPI) anchor assembly. This results in a number of proteins being absent from the cell membrane and altered cellular properties. The affected gene, PIG-A, has been identified and characterized as an enzyme which is responsible for the first step in the synthesis of the GPI anchor; specifically, the synthesis of N-acetyl glucosamine phosphatidyl inositol. This gene has been localized to the X chromosome. The applicant has previously shown that most immature stem cell of patients with PNH lack the activity necessary to allow formation of GPI anchored proteins. He has also cloned both the CDNA for the mouse PIG-A gene and the genomic PIG-A DNA. He proposes to develop an experimental mouse model for PNH, and to use this model to study the biology of the affected stem cells in marrow and in vitro, and to study the effects of various growth factors, cytokines and therapeutic manipulations on the mice who have this abnormal stem cell in their bone marrow. The specific aims include introducing a PIG-A targeted construct into mouse embryonic stem cells to use the PIG-A defective cells in vitro assays permitting analysis of the sequential steps of hematopoietic cell differentiation involving the use of ES cells in vitro and to prepare PIG-A knock-out mouse and employ the chimeric founder mice in back crosses and as a source of PIG-A- hematopoietic stem cells to develop an in vivo model of PNH. He plans to use this model of PNH to study competition between affected and unaffected progenitors in PNH marrow, the in vitro behavior of affected hematopoietic progenitors in long-term marrow cultures, the affects of myelosuppresive agents, cytokines and growth factors on in vivo affected and unaffected stem cell growth, the susceptibility of affected progenitors to transformation, the feasibility of using unaffected progenitors for autologous marrow transplantation following marrow ablation to remove the affected clone, and the feasibility of correcting the PIG-A defect in affected progenitors by gene transfer. These in vitro and in vivo studies will, hopefully, provide new insights into the stem cell physiology in PNH and potential therapeutic approaches for patients with this disorder. In addition, it might provide new information on the relationship of PNH to both aplastic anemia and AML transformation.